1. Field of the Invention
This invention relates to an S. aureus specific intramammary infection (mastitis) assay.
2. Information Disclosure Statement
Staphylococcus aureus is an important pathogen of humans and animals. In dairy cattle, it is the most frequent cause of mastitis which is the most costly disease of food producing animals in the United States. S. aureus persists in infected cows and usually cannot be eradicated from the mammary gland by antimicrobial therapy. Therefore, persistently infected cows are important reservoirs and shedding of the organism contributes to the spread of infection to other cows.
Bovine mastitis is an inflammation of the bovine mammary gland or udder. While mastitis is most often caused by Staphylococcus aureus, it is also attributable to many other organisms including Streptococcus agalactiae, Pseudomonas spp., certain coliform bacteria and mycoplasmas. Mastitis damages the udder and lowers milk production, and therefore imposes an economic burden on the dairy industry. In view of the complex etiology of mastitis, the optimum treatment course may not be apparent until the organism is identified. This is typically done commercially by culturing the organism and classifying it by conventional taxonomic procedures.
Staphylococcus aureus is extremely complex from an immunological standpoint, and a variety of staphylococcal antigens have been studied as potential reagents in immunoassays for staphylococcal antibodies in milk or sera. These include antibodies against protein A, see Live and Ranu, J. Bacteriol., 96:14-23 (1968); enterotoxins, see Fey, et al., J. Clin. Microbiol., 19:34-38 (1984); hemolysins, see Spencer, et al., Am. J. Vet. Res., 24:83-98 (1963); Surujballi and Fackrell, J. Clin. Microbiol., 19:394-98 (1984); Opdebeeck, et al., Am. J. Vet. Res., 43:1770-75 (1982); Christensson, et al., Acta Path. Microbiol. Immunol. Scand., Sec B, 91:351-56 (1983); crude capsular antigens, see Opdebeeck and Norcross, Am. J. Vet. Res., 46:1561 (1985); Watson and Davies, Res. Vet. Sci. 1985, 39:52-58; whole bacteria, see Mathison, et al., Am. J. Vet. Res., 45:2518-24 (1984); teichoic acid, see Granstrom., J. Am. Microbiol., 17:640-46 (1983); peptidoglycan, see Christensson, et al., J. Clin. Microbiol., 19:680-86 (1984 ); leukocidin, See Loeffler and Norcross, Am. J. Vet. Res., 46:1728 (1985); and nucleases, see Gudding, Acta. Vet. Scand., 21:1-14 (1980).
Human patients with deep-seated S. aureus infections have serum antibodies to staphylococcal antigens which can be used for diagnostic purposes. Several tests using different antigens and test formats are described, the most common test being the detection of antibody to S. aureus specific teichoic acid.
Mammary gland secretions of the cow contain immunoglobulins of blood and local origin that may have diagnostic potential. Further, a number of workers have shown that active infection of the mammary gland by S. aureus and/or immunization with S. aureus and/or immunization with S. aureus antigens induce specific immunoglobulins detectable in blood and milk.
Norcross and Obdebeeck, U.S. Pat. No. 4,425,330, used Staphylococcus aureus strain Wood 46 to produce a staphylococcal alpha hemolysin, which they crudely purified by the method of Coulter, J. Bacteriol., 92:1655-62 (1966). This preparation was then used as an ELISA reagent. Similar use was made of "staphylococcal somatic antigen", essentially a crude sonicate of the cells.
Our invention is distinguished from that of Norcross and Obdebeeck by the fact that it uses highly purified antigens with a limited molecular weight range, preferably 18,000 to 26,000 daltons. The significance of their use in an immunological reagent is that virtually all S. aureus infected cows have antibodies in their milk which bind these antigens. Such antibodies are lacking in the milk of uninfected cows. The antigen preparation does not contain alpha or beta hemolytic activity or significant quantities of polysaccharide.
Immunoassays have been developed for the detection of antibodies to specific Staphylococcus aureus antigens. Christensson, et al., Acta Path. Microbiol. Immunol. Scand., Sect. B, 91:351-356 (1983) (alpha and beta hemolysin); Loeffler and Norcross, Am. J. Vet. Res., 46:1728-32 (1985) (leukocidin); Gudding (1980), supra, (nucleases).
Gudding (1980), supra, discloses use of Staphylococcus aureus nuclease in immunoassays of milk and serum. An allegedly statistically significant difference was found between the titres of S. aureus antinucleases in the milk and serum of cows diagnosed as having clinical S. aureus mastitis and the titre in cows which had not been so diagnosed. There was no significant difference between cows diagnosed as having "Staphylococcus aureus latent infection" and those diagnosed as having "infectious mastitis" caused by other microorganisms.
Over 90% of the infected cows examined by the assay of the present invention for immunological reaction between their milk and the disclosed antigenic preparation were subclinical, that is, they showed no visible signs of clinical mastitis, even though their milk exhibited the presence of Staph. aureus when cultured. The assay of Gudding, et al. (1980), unlike our own assay, failed to distinguish subclinical Staph. aureus mastitis ("latent infection") and mastitis caused by other microorganisms. As seen in Gudding Table 2 the titer for subclinical cases averaged 8.7, and for non-Staph. mastitis, 8.6. Gudding speculated that his ability to detect clinical Staph. mastitis was a result of epithelial breakdown, permitting increased transfer of antibody molecules from blood to milk (page 251).
The main commercial method for detecting staphylococcal mastitis is bacterial culture, which has several disadvantages. It detects only live bacteria and therefore antibiotic residues may interfere with detection. It is often inaccurate because of contamination and, therefore, requires that a sterile sample be obtained. It is labor intensive and time consuming. It costs about 10 times as much per sample as the present invention.
The molecular weights of some of the staphylococcal protein antigens are as follows: alpha hemolysin (36,000), beta hemolysin (33,000), gamma hemolysin (45,000) leucocidin (31,000), enterotoxin A (34,700); enterotoxin B (28,366), enterotoxin C (34,100), enterotoxin C.sup.2 (34,000), enterotoxin E (29,600), enterotoxin F (20,000) and protein A (41,000). See Mollby, in Staphylococci and Staphylococcal Infections, at 644-645 (Easman and Adlam, eds: 1983), Vol. 2.
The industry lacks an enzyme-linked immunosorbent assay (ELISA) and method for detecting specific antibodies to a purified antigen fraction of S. aureus.